1. Field of the Invention
The present invention relates to a liquid discharge apparatus for producing a probe carrier having probes of plural kinds in different positions on a carrier, an apparatus for producing a probe carrier utilizing such liquid discharge apparatus, and a method for producing a probe carrier utilizing the same.
2. Related Background Art
In analyzing the base arrangement in a gene DNA or executing genetic diagnosis simultaneously on multiple items with high reliability, it is required to select the DNA of a desired base arrangement with plural probes. As means for providing the probes of plural kinds to be used for such selecting operation, so-called DNA microchip is attracting attention. Also in high throughput screening of pharmaceuticals or in combinatorial chemistry, it is required to execute systematic screening by arranging proteins or chemical solutions of plural kinds (for example 96, 384 or 1536 kinds). There are being developed methods for arraying chemical compounds of plural kinds for this purpose, automated screening technologies in such state, exclusively designed apparatuses therefor and softwares for controlling the screening operations and statistically processing the results.
Such parallel screening operation basically consists of applying so-called probe array, composed of an array of a plurality of known probes constituting selecting means, to a substance to be evaluated, thereby detecting the presence or absence of interaction or reaction with the probes under the same condition. In general, the nature of probes of which interaction or reaction is to be utilized is determined in advance, and, the probes to be mounted on a single probe array are consequently of one substance in general classification, for example a group of DNA probes with different base arrangements. For example, the substances to be utilized in a group of probes are DNA, proteins, synthesized chemical substances (pharmaceuticals) etc. In most cases, there is utilized the probe array containing a group of probes of plural kinds, but, in certain screening operations, there is utilized an array containing plural spots of DNA of the same base arrangement, protein of the same amino acid arrangement or the same chemical substance. Such array is principally utilized for example in the pharmaceutical screening.
More specifically, the probe array containing a group of probes of plural kinds usually assumes a form of arranging, on a substrate, a group of DNA""s having different base arrangements, a group of proteins having different amino acid arrangements or a group of different chemical substances in an array form of a predetermined sequence of arrangement. Among such probe arrays, the DNA probe array is utilized in analyzing the base arrangement of the genetic DNA or in executing the genetic diagnosis simultaneously on multiple items with high reliability.
One of the issues in producing such probe array containing a group of probes of plural kinds is to amount the probes of as many kinds as possible, for example DNA probes of as many different base arrangements as possible, on a substrate. Stated differently, it is necessary to arrange the probes in as high density as possible.
Among the methods for fixing the probes of plural kinds in an array form on the substrate, the U.S. Pat. No. 5,424,186 discloses a method of preparing DNA probes of mutually different base arrangements in an array form, by successive DNA extending reactions utilizing photodecomposable protective radicals and photolithography on a carrier. Such method allows to prepare, for example, a DNA probe array bearing DNA probes of different base arrangements at a density of 10,000 kinds/cm2 or even larger. In this method, the DNA probes of desired base arrangements are synthesized in the predetermined positions on the substrate, through the successive extending reactions, by executing a photolithographic step with an exclusive photomask for each of four bases (A, T, C and G) thereby selectively extending such bases in predetermined positions of the array. Therefore, the cost and time required for preparation increase as the DNA probe chain becomes longer. Also the proportion of the DNA probes including defects in the designed base arrangement is not small because the efficiency of nucleotide synthesis is not 100% in each extending step. Furthermore, in the finally obtained array, the proportion of the DNA probes having the designed base arrangement becomes inevitably smaller since the efficiency of synthesis is lower in the process utilizing the photodecomposable protective radicals in comparison with the ordinary process utilizing acid-decomposable protective radicals.
Furthermore, as the products synthesized directly on the carrier are to be used for the screening operation, it is naturally impossible to eliminate, from the DNA probes having designed base arrangements, those having defects in the base arrangement by a purification process. This method is also associated with a drawback that the base arrangement of the DNA probe synthesized on the substrate cannot be confirmed in the finally obtained array. This means that, in case the extension of a base is scarcely achieved in an extending step for example by an error in the process and the entire probe array becomes defective, the screening operation utilizing such defective probe array results in an erroneous result and there is no way of preventing such situation. The fact that the base arrangement cannot be confirmed is the largest and fundamental drawback of this method.
There is also proposed another method of preparing the probe array, by synthesizing and purifying the DNA for probe in advance, and, eventually after confirmation of the base length thereof, supplying each DNA onto the substrate by a suitable device such as a microdispenser. The PCT laid-open publication WO95/35505 discloses a method of supplying DNA with a capillary onto a membrane. This method in principle allows to prepare the DNA array bearing DNA probes at a density of about 1000 probes/cm2. Basically this method is to prepare the probe array by supplying solution of each probe with a capillary-shaped dispensing device to a predetermined position on the substrate and repeating such operation. This method can be satisfactorily executed if an exclusive capillary is employed for each probe, but, if the number of the available capillaries is limited, it is necessary to sufficiently wash the capillary in changing the kind of probe in order to avoid mutual contamination. It is also necessary to control the supplying position at each operation. Consequently, this method is not suitable for preparing an array bearing probes of many kinds at a high density. In addition, the reproducibility and reliability are not perfect since the supply of the probe solution onto the substrate is achieved by tapping on the substrate with the end of the capillary.
There is also commercially available the microdispenser device, such as HYDRA (trade name) from Robbins Scientific Corp., for feeding solutions of different chemicals to the wells of a microplate of 96 or 384 wells, commonly utilized for the high throughput screening of pharmaceuticals. Such device basically consists of a two-dimensional array of microsyringes with a minimum discharge amount of 100 nl. If such device is applied for the array preparation, the density of the probes will be limited by such minimum discharge amount.
There is also proposed a method, in solid-phase synthesis of DNA on the substrate, of supplying the substrate with a solution of the substance required for synthesis by ink jet method in each extending step. For example, EP publication EPO 703 825B1 discloses a method of executing solid-phase synthesis of plural DNAs of respectively predetermined base arrangements by supplying a nucleotide monomer and an activator, to be utilized in the solid-phase DNA synthesis, from separate piezo jet nozzles. Such supply (coating) by the ink jet method is more reliable, for example in the reproducibility of the supply amount, in comparison with the solution supply (coating) by the aforementioned capillary, and is therefore suitable for achieving higher density of the probe array as the nozzle structure can be made finer. However, this method, basically relying on the successive DNA extending reactions on the substrate, is still associated with the largest drawback that the base arrangement of the DNA synthesized on the substrate cannot be confirmed, as in the aforementioned method disclosed in the U.S. Pat. No. 5,424,186. Though this method is relieved from the cumbersomeness that a photolithographic step involving the exclusive photomask is required for each extending step, it is still insufficient in providing the predetermined probe in each point, which is the essential condition for the probe array. Also the aforementioned EP publication 0 703 825B1 only describes the method of utilizing a plurality of singly formed piezo jet nozzles, and this method is not suitable for preparing the probe array bearing the probes at a high density in case of employing a limited number of nozzles, as in the aforementioned method utilizing the capillaries.
Also Japanese Patent Application Laid-open No. 11-187900 discloses a method of depositing a liquid containing a probe by a thermal liquid discharge unit as a liquid droplet onto a solid phase, thereby forming a spot containing the probe on the solid phase.
Also, in the biochemistry field, the probe solutions for the high throughput screening are usually prepared on a microplate which is standardized to have 96, 384 or 1536 wells with a standardized pitch between the wells. Therefore, if such microplate is applied to a liquid discharge apparatus in which the pitch of the liquid holding portions is determined by the pitch of the nozzles and does not match the pitch of wells of the microplate, there is also required an additional operation of transferring the liquids to such liquid containing portions arranged at the pitch of the discharge nozzles.
As explained in the foregoing, the known methods for preparing the probe array are still associated with various drawbacks or limitations in efficiently preparing the probe array bearing the probes of multiple kinds at a high density on the substrate. For example, in case of transferring the probe solutions synthesized on a microplate to the liquid holding portions arranged with the pitch of the nozzles in the liquid discharge apparatus with a pipette, such pipette has to be washed sufficiently in changing the kind of the probe, in order to avoid mutual contamination. Also if tubes are employed for supplying the probe solutions to the liquid discharge apparatus, the apparatus itself inevitably becomes complex and bulky if there are provided at least several hundred nozzles.
The present invention is to resolve the drawbacks in such liquid discharge apparatus for preparing the probe carrier, and an object of the present invention is to provide a liquid discharge apparatus for producing a probe carrier, allowing to supply the liquid discharge unit with the probe solutions of multiple kinds without increasing the dimension of the apparatus.
Another object of the present invention is to provide an apparatus for producing a probe carrier, allowing to supply the liquid discharge unit with the probe solutions of multiple kinds without increasing the dimension of the apparatus.
Still another object of the present invention is to provide a method for advantageously producing a probe carrier bearing probes of multiple kinds.
As a result of intensive investigation for attaining the above-mentioned objects, the present inventors have found that the supply of the probe solutions of multiple kinds to the liquid holding portions can be achieved with a simple configuration and the freedom in designing can be expanded with respect to the capacity and positioning of the liquid holding portions, by adopting, in the liquid discharge apparatus, a configuration capable of relaxing the limitation on the pitch of the liquid holding portions by the pitch of the nozzles, such as a configuration where a member in which the liquid discharge nozzles are one- or two-dimensionally arranged and a member in which the liquid holding portions to be respectively adjoined with the nozzles are one- or two-dimensionally arranged are mutually laminated.
The present invention is attained by such finding of the present inventors.
More specifically, according to the present invention, there is provided a liquid discharge apparatus for producing a probe carrier bearing, in different positions on a carrier, probes of plural kinds capable of specifically combining with a target substance, the apparatus comprising:
a liquid discharging portion, the number of which corresponds to the number of kinds of the probes, including;
1) a liquid holding portion for holding a liquid containing the above-mentioned probe;
2) a supply opening for supplying the liquid to the liquid holding portion;
3) a liquid discharging nozzle for discharging the liquid; and
4) a flow path connecting the nozzle with the liquid holding portion;
wherein the liquid discharge apparatus is provided with a nozzle opening arrangement face in which the openings of the nozzles are arranged and a supply opening arrangement face in which the liquid supply openings to the above-mentioned liquid holding portions are arranged, and the pitch of arrangement of the supply openings in the supply opening arrangement face is larger than the pitch of arrangement of the openings of the nozzles in the nozzle opening arrangement face.
The configuration of the present invention relaxes the limitation of the nozzle arrangement on the arrangement of the liquid holding portions, and increases the freedom of designing in the arrangement of the liquid holding portions, thereby allowing to provide a liquid discharge apparatus for producing a probe carrier sufficiently capable of handling the probe solutions of multiple kinds with a simple configuration.
The above-mentioned liquid discharge apparatus preferably has a configuration provided with a member including a pair of mutually opposed faces wherein the nozzle opening arrangement face is provided on one of such two faces and the supply opening arrangement face is provided on the other.
Also the liquid holding portion in the liquid discharge apparatus preferably has such a configuration as partly composed of a through hole penetrating from a face of a plate-shaped member to the other face thereof, wherein an end of the through hole is connected to the above-mentioned nozzle. Also such plate-shaped member preferably has the open end of the through hole on a face opposed to the face connected with the nozzle. Further, the through hole is preferably formed by a photolithographic process.
Also the plate-shaped member is preferably water repellent, on the face thereof where the through holes are opened, in the periphery of each opening. Furthermore, the plate-shaped member preferably has a configuration having, on the face thereof including the openings of the through holes, a groove communicating with the openings.
The liquid discharge apparatus of the present invention can assume a configuration including a liquid discharge energy generating element for liquid discharge from the nozzle. Such liquid discharge energy generating element can be composed of a heater element for generating thermal energy to heat the probe solution thereby inducing film boiling therein and causing the resulting pressure to discharge the probe solution from the nozzle opening (discharge port). Also the liquid discharging portion provided in the liquid discharge apparatus preferably has a configuration where a bubble is generated in the probe solution at the discharge of the probe solution from the nozzle by the activation of the heater element and such generated bubble communicates with the external air through the nozzle. The present invention also includes the combinations of these configurations.
According to the present invention, there is also provided an apparatus for producing a probe carrier bearing, in different positions on a carrier, probes of plural kinds capable of specifically combining with a target substance, the apparatus comprising:
A) a liquid discharge apparatus including a liquid discharging portion, the number of which corresponds to the number of kinds of the probes, provided with;
1) a liquid holding portion for holding a liquid containing the above-mentioned probe;
2) a supply opening for supplying the liquid to the liquid holding portion;
3) a liquid discharging nozzle for discharging the liquid; and
4) a flow path connecting the nozzle with the liquid holding portion;
wherein the liquid discharge apparatus is provided with a nozzle opening arrangement face in which the openings of the nozzles are arranged and a supply opening arrangement face in which the liquid supply openings to the above-mentioned liquid holding portions are arranged, and the pitch of arrangement of the supply openings in the supply opening arrangement face is larger than the pitch of arrangement of the openings of the nozzles in the nozzle opening arrangement face; and
B) aligning means for aligning the relative position of the carrier and the liquid discharge apparatus.
The configuration of the present invention relaxes the limitation by the nozzle arrangement on the arrangement of the liquid holding portions, and increases the freedom of designing in the arrangement of the liquid holding portions, thereby allowing to provide an apparatus sufficiently for producing a probe carrier capable of handling the probe solutions of multiple kinds with a simple configuration.
According to the present invention, there is also provided a method for producing a probe carrier bearing, in different positions on a carrier, probes of plural kinds capable of specifically combining with a target substance, the method comprising:
A) a step of preparing a liquid discharge apparatus including a liquid discharging portion, the number of which corresponds to at least the number of kinds of the probes, provided with;
1) a liquid holding portion for holding a liquid containing the above-mentioned probe;
2) a supply opening for supplying the liquid to the liquid holding portion;
3) a liquid discharging nozzle for discharging the liquid; and
4) a flow path connecting the nozzle with the liquid holding portion;
wherein the liquid discharge apparatus is provided with a nozzle opening arrangement face in which the openings of the nozzles are arranged and a supply opening arrangement face in which the liquid supply openings to the above-mentioned liquid holding portions are arranged, and the pitch of arrangement of the supply openings in the supply opening arrangement face is larger than the pitch of arrangement of the openings of the nozzles in the nozzle opening arrangement face; and
B) a step of discharging the liquid containing the probe onto the different positions on the carrier from the liquid discharge apparatus.
The present invention allows to provide a method of advantageously producing a probe carrier bearing probes of multiple kinds.